The failure of the spinal cord to regenerate can be attributed both to a lack of trophic support for regenerating axons and to upregulation of inhibitory factors such as chondroitin sulphate proteoglycans including NG2 following injury. Lentiviral vector-mediated gene therapy is a possible strategy for treating spinal cord injury (SCI). This study investigated the effect of lentiviral vectors expressing Neurotrophin-3 (NT-3) and short-hairpin RNA against NG2 (NG2 sh) to enhance neurite outgrowth in in vitro and ex vivo transection injury models. Conditioned medium from cells transduced with NT-3 or shNG2 lentiviruses caused a significant increase in neurite length of primary dorsal root ganglia neurons compared to the control group in vitro. In an ex vivo organotypic slice culture (OSC) transduction with Lenti-NT-3 promoted axonal growth. Transducing OSCs with a combination of Lenti-NT-3/NG2 sh lead to a further increase in axonal growth but only in injured slices and only within the region adjacent to the site of injury. These findings suggest that the combination of lentiviral NT-3 and NG2 sh reduced NG2 levels and provided a more favourable microenvironment for neuronal regeneration after SCI. This study also shows that OSCs may be a useful platform for studying glial scarring and potential SCI treatments.